Method for the selective determination of procalcitonin 1-116

ABSTRACT

The present invention provides an immunodiagnostic method for determining procalcitonin and procalcitonin derivatives in a biological sample of a patient, in particular in the monitoring and control of treatment and the monitoring of the progression of a local or systemic bacterial infection, inflammation, sepsis or neurodegenerative disease. In particular, the method detects molecular forms of intact procalcitonin 1-116, or procalcitonin partial peptides derived therefrom that retain amino acids alanine and proline (Ala-Pro, AP) in positions 1 and 2 of the amino terminus of the complete procalcitonin 1-116. The method uses antibodies that selectively bind an epitope comprising amino acids 1 and 2 of procalcitonin 1-116 (SEQ ID NO: 1) and can distinguish between intact procalcitonin 1-116 and for example, procalcitonin 3-116.

The invention relates to the area of medical diagnosis, in particularthe determination of biomarkers in biological fluids by means ofimmunodiagnostic methods. More precisely, the invention relates to thedetermination of the peptide procalcitonin for diagnostic purposes, aselective measured value not obtainable to date being determined for thecomplete procalcitonin 1-116 and being used in particular for a moresensitive early diagnosis, monitoring of treatment and monitoring of theprogression of systemic and local infections, in particular of bacterialinfections and of sepsis.

In the context of the present invention, terms such as “diagnosis” or“for diagnostic purposes” are used as a general term for medicaldeterminations which may arise out of different problems depending onthe clinical condition of the patient from which the determination iscarried out and which serve in particular for the diagnosis and earlydiagnosis, the determination of severity and the assessment of theprogression, including the treatment-accompanying assessment of theprogression, and the prognosis of the future progression of a diseaseand the risk stratification of patients. Even if, in the followingdescription, particular attention is made to improved monitoring ofprogression and treatment, no limitation of the term “for diagnosticpurposes” to such specific diagnostic aims is associated therewith.

The mature prohormone procalcitonin (PCT) is a peptide which consists of116 amino acids and was first discussed as precursor of the importanthormone calcitonin (thyreocalcitonin) and the complete amino acidsequence of which (SEQ ID No: 1) has long been known, as have thedetails of its normal proteolytic degradation, which leads to theliberation of the mature hormone calcitonin and other shorter peptides,including in particular so-called katacalcin (procalcitonin 96-116) andan N-terminal peptide (N-procalcitonin 1-57), which are designatedherein, inter alia, as “PCT partial peptides”. As explained moreexactly, for example, in the patents EP 0 656 121 B1 or U.S. Pat. No.5,639,617 of the Applicant and in publications such as ASSICOT M.,GENDREL D., CARSIN H., RAYMOND J., GUILBAUD J., BOHUON C. (1993): Highserum Procalcitonin concentrations in patients with sepsis andinfection. Lancet 341:515-518, severe bacterial infections and certainparasitic and fungal infections with a systemic inflammatory reactioninduce the liberation of PCT in the circulation, where it is found invery high, readily measurable amounts (cf for example also the overviewarticles in O'CONNOR E., VENKATESH B., LIPMAN J., MASHONGONYIKA C., HALLJ. (2001): Procalcitonin in critical illness. Critical Care andResuscitation 3:236-243; WHICHER I., BIENVENU J., MONNERET G. (2001):Procalcitonin as an acute phase marker. Annals of Clinical Biochemistry38:483-893; BECKER K. L., NYLEN E. S., WHITE J. C., MUELLER B., SNIDERR. H. (2004): Procalcitonin and the calcitonin gene family of peptidesin inflammation, infection and sepsis: a journey from procalcitonin backto its precursors. Journal of Clinical Endocrinology and Metabolism 89:1512-1525). Viral, autoimmune and allergic diseases on the other hand donot lead to a significant increase in the measurable PCT concentrationin the blood. PCT reflects the severity of a bacterial infection and isused as a marker for the diagnosis and the therapeutic monitoring ofsepsis, severe sepsis and septic shock of bacterial origin (DANDONA P.,NIX D., WILSON M. F., ALJADA A., LOVE J., ASSICOT M., BOHUON C. (1994):Procalcitonin increase after endotoxin injection in normal subjects.Journal of Clinical Endocrinology and Metabolism 79:1605-1608; GENDRELD., ASSICOT M., RAYMOND J., MOULIN F., FRANCOUAL C., BADOUAL J., BOHOUNC. (1996): Procalcitonin as a marker for the early diagnosis of neonatalinfection. Journal of Paediatrics 128:570-573; SNIDER R. H. JR., NYLENE. S., BECKER K. L. (1997): Procalcitonin and its component peptides insystemic inflammation: immunochemical characterization. Journal ofInvestigative Medicine 45:552-560; WHANG K. T., STEINWALD P. M., WHITEJ. C., NYLEN E. S., SNIDER R. H., SIMON G. L., GOLDBERG R. L., BECKER K.L. (1998): Serum calcitonin precursors in sepsis and systemicinflammation. Journal of Clinical Endocrinology and Metabolism83:3296-3301; MUELLER B., BECKER K. L., SCHACHINGER H., RICKENBACHER P.R., HUBER P. R., ZIMMERLI W., RITZ R. (2000): Calcitonin precursors arereliable markers of sepsis in a medical intensive care unit. CriticalCare Medicine 28:977-983). The general technical knowledge recorded insaid patents and literature references is expressly referred to forsupplementing the present description.

The determination of PCT can also be used for differential diagnosticpurposes since, on the basis of the measurable PCT concentrations inserum and plasma, it is possible to distinguish inflammatory diseasesbased on bacterial infections from those of non-infectious aetiology(cf. also EP 0 880 702 B1). This possibility of distinguishing hasproved to be very valuable for controlling the use of antibiotics in thetreatment, in that it is possible to avoid administering antibioticsalso in cases where they are not effective because the patient does nothave a bacterial infection.

Attempts have also already been made to determine procalcitonin in thecase of infections in the cerebrospinal fluid (CSF). In all casesdescribed in the literature, a commercial assay developed for sepsisdiagnosis and having a functional assay sensitivity (FAS) of only 300ng/l was employed. The measured results obtained were contradictory. Asdescribed in the patent application DE 10 2005 034 174.8 just publishedor in WO 2007/009789, however, more significant measured results can beobtained if a more sensitive assay is employed, which has also beenavailable for some time. With such an assay, a significant correlationof the measurable PCT immunoreactivity with the severity of the diseaseis obtained even in the case of neurodegenerative diseases. Even if thedetermination of PCT in CSF is not specifically discussed below, it isexpressly within the scope of the present invention to use the methodaccording to the invention which is described herein also in thispartial area of diagnostics.

A determination in urine is in principle also covered by the presentinvention.

The determination of PCT is effected, as described in theabove-mentioned patents and literature references, in a suitable mannerby immunoassays of the sandwich type with the use of two antibodieswhich bind to the amino acid sequence of the complete PCT peptide sothat the PCT processed completely with liberation of calcitonin is notdetected but the total unprocessed PCT and optionally also those longerPCT partial peptides which still have both binding sites for the twoantibodies used in a sandwich assay within a single molecule aredetected, it not being possible to distinguish between optionallyterminally modified PCT forms. Conventional methods employ twoantibodies which in general bind to those segments of the complete PCTpeptide which, on proteolytic processing of PCT with formation ofcalcitonin, come to be located on different PCT partial peptides amongthe PCT partial peptides formed or which are located on PCT partialpeptides which do not comprise the calcitonin sequence.

The fact that the complete PCT 1-116 (SEQ ID no: 1) is not found or atleast not primarily found in serum or plasma in the case of sepsis butinstead a PCT 3-116 which is shorter by two amino acids (SEQ ID no: 2)is explained in EP 1 121 600 A1 or EP 1 408 334 A1 or U.S. Pat. No.6,756,483, which is referred to for supplementing the presentdescription. Furthermore, in this context, reference is made to thepublication: WEGLOHNER W, STRUCK J, FISCHER-SCHULZ C, MORGENTHALER NG,OTTO A, BOHUON C, BERGMANN A: Isolation and characterization of serumprocalcitonin from patients with sepsis. Peptides 2001; 22: 2099-103. Asexplained in said publications, the only biomolecule with procalcitoninimmunoreactivity which was characterizable in the investigated samplesfrom sepsis patients was procalcitonin 3-116 mentioned (SEQ ID no: 2).In the further work: WRENGER S, KAHNE T, BOHUON C, WEGLÖHNER W, ANSORGES, REINHOLD D: Amino-terminal truncation of procalcitonin, a marker forsystemic bacterial infections, by dipeptidyl peptidase IV (DP IV); FEBSLett 2000; 466:155-9, it is shown that this procalcitonin 3-116 can beobtained from a synthetically prepared procalcitonin 1-116 by the actionof dipeptidyl-aminopeptidase IV (DAP IV; DP IV; CD 26; E.C. 3.4.14.5).

None of said publications reveals whether the procalcitonin 3-116characterized in the case of sepsis and infections is directly secretedor whether it is subsequently formed proteolytically from a “complete”precursor in the form of procalcitonin 1-116 (PCT 1-116) released as anintermediate into the circulation. Said publications reveal just aslittle regarding whether a “complete procalcitonin” 1-116 occurs inmeasurable steady-state concentrations in the biological samplesinvestigated (whole blood or serum or plasma) or whether it makes asignificant contribution to the measured signal obtained in theconventional procalcitonin determination.

For the determination of the procalcitonin immunoreactivities inserum/plasma, there are various commercial assays of the Applicant, e.g.the chemiluminescence assay B.R.A.H.M.S PCT LIA (B.R.A.H.M.S AG), whichhas an FAS of about 300 ng/l and is tailored to PCT determination insepsis, where very high PCT concentrations may occur. For PCTdetermination with higher sensitivity, a modified sandwich immunoassaywas recently developed which operates with an affinity-purifiedpolyclonal antibody and which is described in more detail inMORGENTHALER N. G., STRUCK J., FISCHER-SCHULZ C., BERGMANN A. (2002):Sensitive immunoluminometric assay for the detection of procalcitonin.Clinical Chemistry 48: 788-789, and is available as B.R.A.H.M.SPCTsensitiv LIA (B.R.A.H.M.S AG). This assay has a substantially betterfunctional assay sensitivity (FAS) of 7 ng/l. With the aid of thisassay, it was possible to determine a mean PCT serum concentration of13.5 ng/l (13.5 pg/ml) in healthy persons, values from <7 to 63 ng/lhaving been found and the 97.5% percentile being at 42.5 ng/l.

In order to enable the hospital physician to carry out a rapid check ofthe status of a potential sepsis patient directly in hospital(“point-of-care”), a one-step fast test in the form of animmunochromatographic assay for rapid semi-quantitative determination ofprocalcitonin in human serum or human plasma of a patient is available(PCT-Q; B.R.A.H.M.S Aktiengesellschaft).

As is evident, for example, from the publications and overview articlescited above, the determination of the biomarker procalcitonin is nowfirmly established in chemical routine, primarily for early diagnosis ofsepsis. A reason for this success is a reliable correlation betweensepsis or bacterial infections and an increase in the measurable PCTimmunoreactivity in the blood or serum/plasma of the patients, andrelatively simple measurability of this PCT immunoreactivity owing tothe high concentrations which are reached in the blood of sepsispatients, due to the high stability of the PCT 3-116 at least primarilydetected in the measurement.

In spite of these indisputable advantages of the determination of thePCT immunoreactivity by the assays available to date for this purpose,however, the inventors discovered that the high stability and the highconcentrations of the measured PCT immunoreactivity are also associatedwith certain disadvantages for certain clinically relevant diagnosticproblems:

A high stability or “long life” of an analyte, e.g. of a peptide such asPCT 3-116, means that the measurement of such an analyte providessummary information about the already elapsed secretory activities for arelatively long period. If, for example, more or less continuousproduction of an analyte during an acute pathological process isassumed, the stable analyte accumulates in the biological sample, andits past physiological production is reflected cumulatively in itsmeasurable concentrations, but reduced by its concentration reductionwhich has taken place in the same period at its physiological clearancerate (its disappearance from the sample owing to its conversion intodegradation products which are not detectable analytically, itsexcretion via the kidneys or owing to other physiological mechanismsknown per se). The more long-lived an analyte or the lower its clearancerate, the smaller is the influence of the exact measuring time on thedetermination of the abovementioned “formation” or “secretion” of theanalyte. For example, in this picture, a concentration which is constantfor relatively long periods may mean that formation and clearancebalance one another. If the concentration decreases, this may indicatethat the secretion of the analyte, which acts in the sense of“concentration replenishment”, has decreased. Nevertheless, theconcentration of the analyte in the blood may still have high values.If, however, the measurable concentration values are high and theclearance rate low, changes in the secretion of an analyte manifestthemselves only as relatively small, poorly interpretable changes ofrelatively large numbers.

If, however, the clinician is interested precisely in rapidlyestablishing changes in the secretion of the analyte, for examplebecause he wishes to draw conclusions about the success of a treatmentor recurrence or reinfection on the basis of these changes, it isfrequently difficult to recognise such a change rapidly andunambiguously on the basis of the measured concentration values of“long-lived” analytes.

The inventors therefore set themselves the object of improving thedetermination of procalcitonin in the sense that changes of theprocalcitonin secretion can be recognised rapidly and reliably so thatthe periods for recognition of the success of a treatment or theintroduction of necessary therapeutic interventions are shorter and thatit is possible to obtain additional, previously unavailable informationwhich immediately permits conclusions about how far an infection/sepsishas already progressed when the physician has to assess the patient forthe first time.

This object is achieved by a method according to claims 1 to 12 and bythe provision of the novel antibodies according to claims 13 to 15 whichare required for such a method, and the corresponding kits according toclaims 16 to 22.

Further important features of the present invention are evident to theperson skilled in the art from the following description and theexplanations of the examples, in particular from the discussions of themeasured results given, these explanations and examples hereby beingincorporated by reference.

At the beginning of the inventors' considerations, it was assumed thatthe peptide procalcitonin, which is determined as PCT immunoreactivityby the conventional assay methods, is presumably formed from itsassociated prepeptide (preprocalcitonin; pre-PCT) having 141 aminoacids, the amino acid sequence of which is known (SEQ ID no: 3). As isgenerally known for many prohormones, PCT is formed from this longerprecursor molecule by elimination of the signal sequence. Although thecleavage point has not been determined experimentally, signal sequencesgenerally have similar patterns, so that cleavage points can bepredicted with high probability. In the case of pre-PCT, the signalsequence should comprise the amino acids 1 to 24 of pre-PCT (SEQ ID no:3). It follows from this that the amino-terminal amino acid in position1 of the prohormone PCT corresponds to the amino acid 25 of pre-PCT (SEQID no: 3). On the basis of such considerations, it was originallyassumed that the PCT found in the circulation during sepsis comprises116 amino acids after elimination of the signal sequence and has theamino acid sequence PCT 1-116 (SEQ ID no: 1). Since, however, only PCT3-116 (SEQ ID no: 3) could be characterized in samples in sepsispatients, there remained the question of intermediate secretion of PCT1-116 and the possibly achieved concentrations of such a PCT 1-116 inthe circulation. It should once again be pointed out that the assays ofthe sandwich type which have been used to date for determiningprocalcitonin immunoreactivity operate with combinations of twoantibodies which do not permit a distinction between PCT 1-116 and3-116. The known assays therefore cannot of course selectively detectsaid different forms of PCT, and they therefore also do not detectpossible characteristic changes in the relative molar amounts of saidanalytes if such changes occur in the course of a pathological process,for example in the course of a sepsis. The PCT immunoreactivitydetermined by a conventional assay is therefore also referred to as “PCTtotal” in the present application.

The inventors decided in this situation to provide a novel type ofmethod for the immunodiagnostic determination of procalcitonin, by meansof which only those procalcitonin fractions which also have the firsttwo amino acids of procalcitonin 1-116 are selectively determined, and,with the use of this novel method, to check whether it is possibletherewith to obtain clinically relevant measured results which arequalitatively better than the results of the prior determination ofprocalcitonin immunoreactivity.

As described in more detail in the following experimental part, theinventors first developed novel antibodies having the binding propertiesrequired for a clinical determination, so that they recognise only thoseprocalcitonin molecules which contain the complete procalcitoninsequence at the amino terminus with the first two amino acids (Ala-Pro)of procalcitonin 1-116 or which contain at least amino acid 2 (Pro).They then used these novel antibodies in an assay of the sandwich typewhich permits a selective determination of the PCT 1-116 in a biologicalsample of a human patient, for example in serum or in particular inplasma, without the measured value being influenced by the presence ofPCT 3-116 (SEQ ID no: 2) in the sample.

As shown below in the experimental part of the application, the measuredresults obtained using such an assay fulfilled the hopes of theinventors for the desired improvement of the diagnostic determination ofprocalcitonin for the above-mentioned particular diagnostic purposes.

It was in fact found that significant, readily measurable concentrationsof PCT 1-116 are present in fresh samples of human serum and inparticular of fresh human plasma of patients. The molar concentrationsof PCT 1-116 found by the inventors in such samples are in the range ofabout ⅕ to ⅛ of the molar concentrations for the PCT immunoreactivitymeasurable in parallel by traditional methods (“PCT total”).

If it is assumed that the total PCT secreted in the circulation issecreted primarily as PCT 1-116, it follows that the concentrations ofPCT 1-116 and of PCT total must be equal at a hypothetical time at thebeginning of the PCT secretion so that a maximum hypothetical initiallimit of 1.0 can be assumed for the ratio of PCT 1-116 to PCT total.Ratios in the range of about ⅕ to ⅛, as were found by the inventors inthe samples investigated, therefore already contain per se informationabout the history of the sample, for example in the sense of the elapsedduration and/or severity of the infection/sepsis. Owing to the lowerstability of PCT 1-116 (because of the conversion into PCT 3-116), saidratio and the concentration changes which can be established for PCT1-116 reflect the acute status of a patient very much more directly thanthe values for the more stable PCT 3-116. The selective determination ofPCT 1-116 therefore leads to considerable diagnostic progress. This alsomanifests itself in particular when the selective determinationaccording to the invention of the PCT 1-116 with the aid of the novelmethod is combined with the conventional determination of the total PCTimmunoreactivity (PCT total).

The method according to the invention has particular advantages in caseswhere a rapid detection of the actual disease status of a patient isdesirable. In principle, however, it can be used in all cases where theknown methods have been used to date for determining the PCTimmunoreactivity. As long as a determination is selective for PCT 1-116with both amino-terminal amino acids (Ala-Pro) or at least for aprocalcitonin with the amino acid in position 2 (Pro), the method is tobe considered as one according to the present invention. The specialassay form chosen plays a fairly minor role, and a special assay formatis chosen primarily from practical points of view relating tomeasurement technology.

Although a specific method of the sandwich type with the use of specificantibodies (monoclonal antibodies) is therefore described in theexperimental part of this application and used for the measurement, thepresent invention is not limited to this specific method or a method ofa similar type. Rather, it includes any desired immunodiagnostic methodswhich, in the context of the present invention, permit a selectivedetermination of PCT 1-116 without the measured value being influencedby simultaneously present other PCT derivatives, in particular PCT3-116.

It is self-evident to the person skilled in the art that, for example,assays of the competitive type (for example with an immobilizedselective antibody and a marked/selectively markable analogue of theanalyte which competes with PCT 1-116; or with an immobilized analogueand a marked selective antibody) or other assay formats, for exampleso-called turbidometric assays, can also be used instead of the assay ofthe sandwich type described.

In principle, any known marker suitable for diagnostic assays can beused (i.e. instead of the luminescence marking used in the examples, forexample, radioisotopes, enzymes, fluorescence labels, otherchemoluminescence labels or bioluminescence labels and directlyoptically detectable colour markings, such as, for example, gold atomsand dye particles, as used in accelerated tests, can also be used).

It is furthermore expressly within the scope of the present invention todesign the method according to the invention as an accelerated test, forexample in the form of an immunochromatographic assay method.

If the method is designed as a heterogeneous method in which a specificbinder in immobilized form is present bound to a solid phase and/or thereaction products are at least partly immobilized on a solid phase, thesolid phase may be any desired suitable solid phase, for example a wallof a test tube, a particulate solid phase, for example in the form ofmagnetic particles suspended in the reaction solution, or a solid phasein the form of a support of an immunochromatographic apparatus (for anaccelerated test).

In a currently preferred embodiment, the method is carried out as aheterogeneous sandwich immunoassay in which one of the antibodies isimmobilized on the walls of coated test tubes (e.g. of polystyrene;“coated tubes”; CT) or on microtitre plates, for example of polystyrene,or on particles, for example magnetic particles, while the otherantibody carries a residue which represents a directly detectable labelor permits a selective link with a label and serves for detection of thesandwich structures formed. Delayed or subsequent immobilization withthe use of suitable solid phases is also possible.

The method need not however be designed as a heterogeneous method inwhich at least one specific binder is present in immobilized form. Allreactants and reaction products can also be present suspended in ahomogeneous liquid phase and can remain there for the measurement. Insuch a case it is preferable to mark both antibodies used for thedetermination with parts of a detection system which permits signalgeneration or signal triggering when both antibodies are integrated intoa single sandwich. Such techniques can be designed in particular asfluorescence amplification or fluorescence extinction detection methods.A particularly preferred method of this type relates to the use ofdetection reagents to be used in pairs, as described, for example, inU.S. Pat. No. 4,822,733, EP-B1-180 492 or EP-B1-539 477 and the priorart cited therein. They permit a measurement which selectively detectsonly reaction products which contain both marking components in a singleimmune complex, directly in the reaction mixture. As an example,reference may be made to the technology offered under the brands TRACE®(Time Resolved Amplified Cryptate Emission) or KRYPTOR®, which implementthe teachings of the abovementioned applications.

It is furthermore assumed that the assay method according to theinvention can advantageously also be used as part of a so-calledmulti-parameter diagnosis. Further parameters determined thereby are,for example, sepsis and infection parameters which may be selected, forexample, from a group which consists of anti-ganglioside antibodies, theproteins total procalcitonin (PCT total), procalcitonin 3-116, CA 125,CA 19-9, S100B, S100A proteins, LASP-1, soluble cytokeratin fragments,in particular CYFRA 21, TPS and/or soluble cytokeratin-1 fragments(sCY1F), the peptides inflammin and CHP, other peptide prohormones, suchas, in particular, pro-ANP, pro-BNP, pro-adrenomedullin, pro-endothelin,pro-vasopressin and the diagnostically usable peptide fragments thereof,glycine N-acyl transferase (GNAT), carbamoyl phosphate synthetase 1(CPS 1) and the C-reactive protein (CRP), cytokines, e.g. of theinterferon type, or suitable fragments thereof. In said multi-parameterdeterminations, it may be intended to determine the measured results fora plurality of parameters simultaneously or in parallel and to evaluatethem, for example, with the aid of a computer program which also usesdiagnostically significant parameter correlations.

A particularly important combination measurement is a parallel selectivemeasurement of PCT 1-116 and the customary PCT immunoreactivity (PCTtotal), which, in the light of the results of the present application,represents a summary measurement of the concentrations of PCT 1-116 plusPCT 3-116 present in a sample. Regarding the advantages of such ameasurement, reference is expressly made to the following examples.

It is furthermore possible to combine selective measurements of PCT1-116 and PCT 3-116. The inventors have also carried out suchcombination measurements with the use of a monoclonal antibody selectivefor PCT 3-116 (results not shown). Since the concentrations of PCT 3-116constantly newly formed in the proteolytic degradation of PCT 1-116 aredirectly included in the concentrations of PCT 3-116, the evaluation ofthe comparative measurements is, however, more complex. In addition, itappears that a further proteolytic degradation may occur at the aminoterminus of PCT 3-116, with the result that the measured results arefalsified. A combination measurement of PCT 1-116 with a selectivemeasurement of PCT 3-116 is therefore currently less preferable.

In the following examples, reference is made to figures in which:

FIG. 1 shows a standard curve of a sandwich assay for the selectivedetermination of PCT 1-116, as described in more detail in theexperimental part;

FIG. 2 shows a standard curve of a conventional B.R.A.H.M.S PCT LIAafter adaptation of the calibration curve to the unit pmol/l;

FIG. 3 shows comparative measurements of the change in the concentrationof PCT 1-116 by the assay according to the invention (triangles) and theconcentration of PCT total by a commercial assay (B.R.A.H.M.S PCT LIA;squares), based in each case on the coordinated starting concentrationson day 0 and measured in EDTA plasmas of eight successfully treatedsepsis patients of an intensive care ward;

FIG. 4 shows results of the determination of the molar ratio of PCT1-116 to PCT total in samples according to FIG. 3 and the change thereofas a function of time in the course of 3 days.

EXPERIMENTAL PART I. Assay Development—Peptide Syntheses and AntibodyDevelopment

I.1. Peptide Syntheses

The following peptides were synthesised as antigen constituents forantibody production, as selective binding partners for affinitypurification and for epitope mapping and as potential standards andcompetitors:

Derived from the known amino acid sequence of pre-procalcitonin(pre-PCT; SEQ ID no: 3), a plurality of partial peptide segments wereselected and were chemically synthesised as soluble peptides by standardmethods. These were purified, subjected to quality control by means ofmass spectrometry and reversed phase HPLC and lyophilized in aliquots(JPT, Berlin, Germany). The amino acid sequences of the peptidessynthesised are (the stated amino acid positions are based on pre-PCT;amino acid 25 corresponds to amino acid 1 of PCT 1-116 according to SEQID no: 1):

PAS10 APFRSALESC 25-33 (SEQ ID NO: 4) (+C-terminal cysteine) PAD21AAPFRSALESSPADPATLSED 24-44 (SEQ ID NO: 5) PAD20 APFRSALESSPADPATLSED25-44 (SEQ ID NO: 6) PPD19 PFRSALESSPADPATLSED 26-44 (SEQ ID NO: 7)PFD18 FRSALESSPADPATLSED 27-44 (SEQ ID NO: 8) PRD17 RSALESSPADPATLSED28-44 (SEQ ID NO: 9) PSD16 SALESSPADPATLSED 29-44 (SEQ ID NO: 10) PAN40APFRSALESSPADPATLSEDMSS 25-44/122-141 (SEQ ID NO: 11) DLERDHRPHVSMPQNAN

I.2. Development, Marking and Characterization of Antibodies withSpecificity for the N-Terminus of PCT 1-116

I.2.1. Development and Selection of Monoclonal Antibodies

Monoclonal antibodies were developed by standard methods, as described,for example, by Harlow and Lane (Harlow E., Lane D. “Antibodies—ALaboratory Manual”, Cold Spring Harbor Laboratory, 1988, ISBN0-87969-3,4-2). The development is summarized in more detail below:

By means of MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), thepeptide PAS10 (SEQ ID NO: 4; amino acids 1 to 10 of PCT 1-116) wasconjugated with the carrier protein KLH (keyhole limpet haemocyanine)(cf operating instructions “NHS-Esters-Maleimide Crosslinkers” fromPIERCE, Rockford, Ill., USA). Balb/c mice were immunized with thisconjugate. After boosting, spleen cells of the mice were subsequentlyfused with SP2/0 myeloma cells.

For binding tests of the antibodies from the culture supernatants of themyeloma cells, the peptides PAD20 (SEQ ID NO: 6 and PPD19 (SEQ ID NO: 7;amino acids 2-20 of PCT 1-116) were immobilised on microtitre plates.For this purpose, the peptides were dissolved in 20 m/M Na phosphate, pH7.4, 50 mM NaCl in a concentration of 7 μg/ml, and 150 μl thereof werepipetted per cavity. Incubation was effected for 20 hours at 4° C. Thesolution was filtered with suction. Each cavity was then filled with 150μl of 10 mM sodium phosphate, 2% Karion FP, 0.3% bovine serum albumin,pH 6.5. After 20 hours the solution was filtered with suction.

150 μl of each of the culture supernatants to be tested were pipettedinto microtitre plate cavities coated with PAD20 (SEQ ID NO: 6) or PPD19(SEQ ID NO: 7). Incubation was effected for two hours at 22° C. Thesupernatant was filtered with suction. Washing was effected twice with20 mM Na phosphate, pH 7.4, 50 mM NaCl, and bound antibodies weredetected with secondary anti-mouse antibody-alkaline phosphataseconjugate by standard methods (SIGMA, Deisenhofen, Germany).

Those cell cultures which secreted antibodies which had bound to thepeptide PAD20 (SEQ ID NO: 6) but far less to the peptide PPD19 (SEQ IDNO: 7) were selected for isolation. Culture supernatants from theisolations were screened by the same method as the initial cellcultures. Cell clones screened positively in this sense were thencultivated on a larger scale, and the antibodies were purified by meansof affinity chromatography on a protein G column, and three of saidantibodies were further investigated as potential candidates for theselective binding of PCT 1-116 in an immunoassay (internal designationsof the antibodies: mAb 295/3H12; mAb 295/4G9; mAb 295/4611).

I.2.2. Marking of the Antibodies

For characterization of the epitope specificity of the three selectedantibodies and subsequent assay development, the monoclonal antibodiesobtained were marked with a chemiluminescence label as follows:

Each antibody was adjusted to a concentration of 1.5 mg/ml in a 100 mMpotassium phosphate buffer (pH 8.0). 67 μl of the antibody solution weremixed with 10 μl of MA70 acridinium NHS ester (1 mg/ml; from HOECHSTBehring) and incubated for 15 minutes at room temperature. Thereafter,423 μl of 1 M glycine were added and incubation was effected for afurther 10 minutes. Thereafter, the marking batch was re-buffered overan NAP-5 gel filtration column (Pharmacia) in 1 ml of mobile phase A (50mM potassium phosphate, 100 mM NaCl, pH 7.4) according to the operatinginstructions and freed thereby from low molecular weight constituents.For separating off the last residues of labels not bound to antibody,gel filtration HPLC was carried out (column: Waters Protein Pak SW300).The sample was applied and was chromatographed at a flow rate of 1ml/min with mobile phase A. The wavelengths 280 nm and 368 nm weremeasured using a flow photometer. The absorption ratio 368 nm/280 nm asa measure of the degree of marking of the antibody was 0.10+/−0.01 atthe peak. The monomeric antibody-containing fractions (retention time8-10 min) were collected and were collected in 3 ml of 100 mM sodiumphosphate, 150 mM NaCl, 5% bovine serum albumin, 0.1% sodium azide, pH7.4.

I.2.2. Epitope Mapping of Three Selected Monoclonal Antibodies

For the epitope mapping of the three developed and selected monoclonalantibodies, various peptides which represent the N-terminal segment ofprocalcitonin (cf. I.1.; Tab. 1) were immobilized on tubes. This waseffected as described above for the coating of microtitre plates, exceptthat 300 μl of peptide solution were pipetted instead of 150 μl.

The respective chemiluminescence-marked monoclonal antibody was dilutedin assay buffer (100 mM sodium phosphate, 150 mM NaCl, 0.5% bovine serumalbumin, 0.1% unspecific mouse IgG, 0.1% sodium azide, pH 7.4), so thatan end concentration of 0.9 million RLU (relative light units)/200 μlwas obtained. 200 μl portions of these solutions were pipetted into thepeptide tubes, and incubation was effected for 2 hours at 22° C. withshaking. This was followed by washing four times with 1 ml portions ofwash solution (0.1% Tween 20) per tube, allowing to drip and measurementof the chemiluminescence bound to the tube in a luminometer (fromBERTHOLD, LB952T; base reagents from BRAHMS AG).

As is evident from the following table 1, all three selected antibodies(internal designation mAb 295/3H12; mAb 295/4G9; mAb 295/4G11) reactedbest with a peptide derived from pre-procalcitonin (or procalcitonin) ifthis ended with position 25 at the N-terminus (peptide PAD20; SEQ ID NO:6). If position 25 was absent (peptide PPD19; SEQ ID NO: 7), onlymarginal binding was achieved, which accounted for only about twice theunspecific binding for the individual monoclonal antibodies and waslower by a factor of 199 (mAb 295/3H12), a factor of 85 (mAb 295/4G9) ora factor of 102 (mAb 295/4G11) than for peptide PAD20 in which position25 was present.

The antibodies are thus highly specific and suitable for distinguishingprocalcitonin peptides which contain positions 1 and 2 of PCT 1-116(positions 25 and 26 of pre-PCT) at the N-terminus from thoseprocalcitonin peptides which are shortened by only one amino acidresidue at the N-terminus in comparison therewith.

TABLE 1 Epitope mapping. Measured binding values are stated as relativeluminescence units (RLU) Amino acid mAb position 295/ mAb mAb PeptideSequence (pre-PCT) 3H12 295/4G9 295/4G11 PAD20 SEQ ID NO: 6 25-44 3618626496 30222 PPD19 SEQ ID NO: 7 26-44 182 310 297 PFD18 SEQ ID NO: 827-44 96 190 171 PRD17 SEQ ID NO: 9 28-44 65 158 164 PSD16 SEQ ID 29-4464 176 146 NO: 10 Control 61 149 162

I.4. Development of a Sandwich Immunoassay for Specific Measurement ofPCT 1-116

I.4.1. Coupling

High-binding 5 ml polystyrene tubes (from Greiner) were coated asfollows with a monoclonal antibody (internal designation: QN05) which isdirected at an epitope in the C-terminal segment of procalcitonin and isalso used in a commercial assay B.R.A.H.M.S PCT LIA: the antibody wasdiluted in 50 mM tris, 100 mM NaCl, pH 7.8, to a concentration of 6.6μg/ml. 300 μl of this solution were pipetted into each tube. The tubeswere incubated for 20 hours at 22° C. The solution was filtered withsuction. Each tube was then filled with 4.2 ml of 10 mM sodiumphosphate, 2% Karion FP, 0.3% bovine serum albumin, pH 6.5. After 20hours, the solution was filtered with suction. Finally, the tubes weredried in a vacuum drier.

I.4.2. Marking

The monoclonal antibody mAb 295/3H12 which is characterized in moredetail above with regard to its binding behaviour and specificallyrecognizes the N-terminus of PCT 1-116 was chemiluminescence-marked asdescribed above under 1.2.2.

I.4.3. Procedure and Evaluation of the Immunoassay

The peptide PAN40 (SEQ ID NO: 11), which was diluted serially in horsenormal serum, served as standard material. Concentrations according tothe peptide weight taken were ascribed to the standards thus prepared.

The sandwich immunoassay was prepared as follows: 50 μl of standards orsamples and 150 μl of assay buffer (100 mM sodium phosphate, 150 mMNaCl, 0.5% bovine serum albumin, 0.1% unspecific mouse IgG, 0.1% sodiumazide, pH 7.4), containing 0.5 million RFU (relative light units) of theMA70-marked antibody, were pipetted into the antibody-coated test tubes.Incubation was effected for 3 hours at 22° C. with shaking. This wasfollowed by washing 4 times with 1 ml portions of wash solution (0.1%Tween 20) per tube, allowing to drip and measurement of thechemiluminescence bound to the tube in a luminometer (from BERTHOLD,LB952T; base reagents from BRAHMS AG). Using the software MultiCalc(Spline Fit), the PCT 1-116 concentrations of the samples were read fromthe standard curve. A typical standard curve is shown in FIG. 1. Thetest had a functional assay sensitivity (FAS), defined as theconcentration at which the coefficient of variation of the mean of tenindependent test runs was 20%, of 6 pmol/l.

Following the above-described experiments of the epitope specificity ofthe antibody 295/3H12, an investigation was carried out to determine theassay cross-reactivity for a peptide which was derived fromprocalcitonin and lacked the first N-terminal amino acid: A sample ofPCT 2-116 (InVivo GmbH, Hennigsdorf, Germany; cf. also EP 1 408 334)having a concentration of 37433 pmol/l was measured as 206 pmol/l. Thecross-reactivity was thus only 0.5%.

II. Measurements on the Analyte Stability and of Human Plasmas of SepsisPatients

II.1. Analyte Stability in Samples

The stability of natural PCT 1-116 in samples from sepsis patients basedon its measurability in the PCT 1-116 assay according to I., was firstinvestigated. For this purpose, EDT plasma and serum samples wereobtained from ten sepsis patients and then measured freshly and afterstorage for six hours at 22° C. by the abovementioned assay.

On average, the decrease in immunoreactivity after 6 h was only 3.6% inthe case of the plasmas, and the largest individual decrease was 7.6%.In the case of the sera, on the other hand, the immunoreactivitydecreased on average by 13.5%, and considerable individual differencesin the decrease were observed.

The observed individual stability differences of PCT 1-116, inparticular in serum, which reflect possibly individually differentenzyme activity, mean that it may not be directly possible to relatemeasurable concentration changes of PCT 1-116 in an individual patientsample to a universally applicable reference value for the eliminationof PCT 1-116 from the circulation, for example in order to distinguishcases of a pure decrease of a fixed starting concentration of PCT 1-116from cases where a persistent or recurring secretion of PCT 1-116 issuperposed on such a concentration decrease.

Procalcitonin, measured as PCT immunoreactivity or “PCT total” by theconventional B.R.A.H.M.S PCT LIA, is stable in serum samples afterstorage for six hours at 22° C. (operating instructions for B.R.A.H.M.SPCT LIA).

The B.R.A.H.M.S PCT LIA is a sandwich immunoassay which uses monoclonalantibodies against epitopes in the C-terminal segment (katacalcinfraction) or mid-regional segment (calcitonin fraction). To permit acomparative consideration, the concentrations of the standards specifiedby the manufacturer in ng/ml were converted into pmol/l. A typicalstandard curve is shown in FIG. 2. The test had a functional assaysensitivity (FAS), defined as the concentration at which the coefficientof variation of the mean of ten independent test runs was 20%, of 20pmol/l.

The determined stability of the analyte PCT 1-116 in human EDTA plasmacan be regarded as being usually sufficient for clinical practice.However, if there is a risk that the respective EDTA plasma obtainedfrom the sample cannot be measured at a sufficiently close time, it iswithin the scope of the present invention to prevent the degradation ofthe PCT 1-116 by dipeptidyl-aminopeptidase IV (DAP IV) to PCT 3-116 byaddition of synthetic inhibitors for DAP IV to the sample (e.g. of Lys[Z(NO₂)]-thiazolidide or of Pro-Pro^((P))[OPh-4Cl]₂; cf. WRENGER S,KAHNE T, BOHUON C, WEGL{umlaut over (O)}HNER W, ANSORGE S, REINHOLD D:Amino-terminal truncation of procalcitonin, a marker for systemicbacterial infections, by dipeptidyl peptidase IV (DP IV), FEBS Lett2000; 466:155-9). If, optionally additionally, other enzymes contributeto the decrease in the concentration of PCT 1-116 in a sample, it is ofcourse also possible to use other inhibitors of the destructive enzymeeffect.

II.2. Measurements on Clinical Value

I.2.1. Kinetics of Successful Treatment of Infections

It is desirable to recognize the success of the treatment in the case ofsepsis patients early and reliably because the safety in the treatmentregime of a patient is increased thereby and expensive stays in theintensive care unit can be reduced.

For checking the question as to whether the measurement, according tothe invention, of PCT 1-116 with the use of the sandwich assay describedabove can give results which differ from those of a conventionalmeasurement of the PCT immunoreactivity (“PCT total”), the followingprocedure was adopted:

Plasma samples were obtained daily from eight sepsis patients who weresuccessfully treated in the intensive care unit. PCT total (measured bythe conventional B.R.A.H.M.S PCT LIA; see above) and PCT 1-116 weremeasured at the beginning of the treatment and on the two subsequentdays. The median concentrations at the beginning of the measurements (onday 0) were 100.6 pmol/l for PCT 1-116 and 591.8 pmol/l for PCT total(molar ratio of the initial concentrations of about 0.17).

The concentrations of PCT 1-116 decreased significantly more rapidlythan those of PCT total: the decrease of PCT 1-116, based on day 0, was32% on the first day and 52% on the second day. In contrast, thedecrease of PCT total was only 11% on the first day and only 30% on thesecond day (FIG. 3).

In view of the diagnostic aim of recognizing the successful treatment asquickly as possible on the basis of a decrease in the procalcitoninsecretion, it is now possible to pose the question concerning the timeafter which the two assay methods compared above enable a decrease to berecognized at all. If inter assay variances are taken into account, itis approximately true, that a decrease of about 20% can be classed asbeing reliably detectable. Such a decrease is detectable after only 0.6day in a measurement of PCT 1-116, but only after 1.2 days in theconventional measurement of the PCT immunoreactivity (of PCT total).

The success of a treatment is therefore substantially more quickly andreliably detectable in the measurement of PCT 1-116 than in theconventional measurement of the PCT immunoreactivity (“PCT total”).

For a further evaluation of the measured data, the respective molarratio of PCT 1-116 to PCT total was calculated for each patient and eachmeasuring time. Over the three days of observation, this ratio,expressed as the median, decreased from 0.17 through 0.15 to 0.12 (FIG.4). This evaluation, too, reflects the different degradation kinetics ofPCT 1-116 and PCT total. Expressed simply, it is true that the decreaseof PCT 1-116 directly reflects the degradation thereof or lowerstability, while the decrease of “PCT total” reflects the degradation ofPCT 3-116 or the stability thereof. Since the conversion of PCT 1-116into PCT 3-116 should not have a substantial effect on the measurementin the determination of PCT total because the conventional method doesnot distinguish between PCT 1-116 and PCT 3-116, the rate of conversionof PCT 1-116 into PCT 3-116 does not significantly influence thedecrease in concentration of PCT total, so that this substantiallycorresponds to the decrease of the more stable PCT 3-116 which hasaccumulated in the sample.

The above considerations show that moreover a small value for the ratioof PCT 1-116 to PCT total (i.e. a low concentration of PCT 1-116 at asimultaneously comparatively high concentration of PCT total) indicatesthe end of the infection, while a large value indicates a time close tothe beginning of the PCT secretion and hence a highly acute infection.This observation has implications not only for observation of theprogression but also for the initial estimation of the infection statusof a patient, for example at emergency admission or on medical treatmentas an outpatient.

II.2.2. Kinetics in the Case of Secondary Infection

During their stay in the intensive care unit, sepsis patients frequentlysuffer a further infection which, owing to the weakened immune system,is particularly life-threatening and can have fatal effects. The earliersuch a secondary infection is detected, the more quickly and effectivelycan therapeutic countermeasures be initiated. Owing to the knownphenomenon of endotoxin tolerance, however, secondary infections resultin a less pronounced increase in the PCT total concentration.

The early detection of a bacterial reinfection which is associated witha less pronounced subsequent increase in the temporarily already reducedor even terminated PCT secretion is particularly complicated in that,owing to the primary infection, the patients already have considerablePCT total concentrations which, due to the high stability of PCT 3-116,are only relatively slowly reduced even with successful treatment. Aswas shown above, PCT 1-116 has substantially faster in vivo degradationkinetics than PCT total (with a predominant proportion of PCT 3-116). Ifit is assumed that PCT 1-116 is converted in the first step of itsdegradation into PCT 3-116, the decrease in the concentration (lower inabsolute values) of PCT 1-116 does not correspond to an increase in thevalues for PCT total. These increase only at the rate of production offresh PCT 1-116.

In the case of an infection which has been substantially withstood andis no longer acute—i.e. before a secondary infection—the measurableconcentrations of PCT 1-116 should be relatively low, although absolutevalues for the concentrations of PCT total are still high owing to thestability of PCT 3-116, which at this time is likely to make the maincontribution to the measured concentration of PCT total. A subsequentincrease in the PCT secretion, induced by a secondary infection, shouldtherefore be detectable at an early stage on the basis of the measuredvalues for PCT 1-116, whereas it is not detectable or not unambiguouslydetectable on the basis of the very much higher measured values for PCTtotal in absolute terms.

The consideration of the PCT 1-116 and PCT 3-116 concentrations measuredtogether in the determination of PCT total leads to the conclusion that,under the influence of a fairly slow decrease of a high concentration ofPCT 3-116 from the primary infection and the subsequent increase of PCTfrom the secondary infection, the beginning of the resecretion of PCT ispossibly detectable not immediately as a measurable increase of thetotal PCT but initially only as a poorly detectable reduction of thedecrease of the PCT total concentrations, which being to decrease moreslowly than corresponds to the clearing rate of PCT 3-116.

An investigation was therefore carried out to determine whether aconcentration development of PCT 1-116 in the sense of an increase orslowing down below the usual stability-related concentration reductionof PCT 1-116, which differs substantially from the concentrationdevelopment of PCT total, predicts a subsequent increase also of PCTtotal on the subsequent day—as a sign of a secondary infection.

Plasma was obtained daily from 44 sepsis patients with an average stayof 10.8 days in the intensive care unit, PCT total and PCT 1-116 weredetermined in parallel in said plasma, and the concentration curvethereof was monitored for each individual patient during the period ofthe measurements.

It was observed six times that an increase of PCT total on thesubsequent day was preceded by an increase of PCT 1-116, while PCT totalstill declined.

In 7 other cases—in each case with continuing decrease of PCT total—areduced reduction of the decrease of the concentration of PCT 1-116,i.e. a slower decrease than in other samples, was observed, which wasassociated with the onset of a secondary infection. The concentrationsof PCT total did not yet change to a significant extent. Only on thesubsequent day were signs of a subsequent increase of PCT totaldetectable. It is within the scope of the present invention also to usethose “hidden” increases of the concentration of PCT 1-116, which arenot yet manifest in the monitoring of PCT total, with the application ofsuitable monitoring and evaluation techniques and mathematical modelssuitable for this purpose in the monitoring of therapeutic sepsispatients for detecting secondary infections.

In summary, it may be said that a PCT 1-116 decrease which is reducedcompared with PCT total or even an increase of PCT 1-116 on thesubsequent day predicts in a highly specific and even sensitive manneran increase of PCT total as an expression of a secondary infection.

In view of the results disclosed above, the inventors contemplate theselective determination of PCT 1-116 according to the method of thepresent invention as alternative to the determination of PCT(total) byan assay recognizing predominantly PCT 3-116. PCT 1-116 can bedetermined in all cases in which the determination of PCT(total) hasalready proved to be useful in the field of medical diagnosis.

In particular, the use of the selective determination of PCT 1-116 forthe diagnosis, differential diagnosis, risk stratification, prognosis,assessment of the course of a disease and the control of therapeuticmeasures is intended or contemplated especially for human patients inconnection with infections, especially infections by gram-negative orgram-positive bacteria and by fungal and/or parasitic etiologicalagents, which infections may cause a locally confined or systemicinflammatory response (for example sepsis, severe sepsis and septicshock), as well as in cases of an underlying co-morbidity in heartfailure patients, and, further, for the purpose of risk stratificationand prognosis of patients suffering from coronary artery disease (CAD).

1. An immunodiagnostic method for determining intact procalcitonin andprocalcitonin derivatives thereof wherein molecular forms ofprocalcitonin, or procalcitonin partial peptides derived therefrom,comprising amino acids alanine and proline (Ala-Pro AP) in positions 1and 2 of the amino terminus of the complete procalcitonin 1-116 (SEQ IDNO: 1) are selectively detected in a biological sample from a patient,but PCT 3-116 (SEQ ID NO:2) is not detected.
 2. The method of claim 1,wherein the determination is carried out with the use of at least oneantibody that specifically binds to a procalcitonin comprising aminoacids in positions 1 and 2 of the complete amino acid sequence ofprocalcitonin 1-116 (SEQ ID NO: 1).
 3. The method of claim 2, whereinthe antibody requires the presence of both amino acids of positions 1and 2 of the complete sequence of procalcitonin 1-116 (SEQ ID NO: 1) forits specific binding.
 4. The method of claim 1, wherein the at least oneantibody is a monoclonal antibody or a polyclonal antibody, for examplea polyclonal antibody purified by affinity chromatography.
 5. The methodof claim 1, wherein the biological sample is a biological fluid which isselected from blood and a blood fraction, in particular serum or plasma,and cerebrospinal fluid (CSF).
 6. The method of claim 1, said method iscarried out as part of the diagnosis of diseases which are selected fromlocal or systemic infections, inflammations, sepsis andneurodegenerative diseases.
 7. The method of claim 6, said method iscarried out in the monitoring and control of the treatment or themonitoring of the progression of one of said diseases.
 8. The method ofclaim 1, wherein said method is designed as an immunochromatographicmethod for point-of-care diagnosis or as another accelerated test. 9.The method of claim 1, said method is carried out as part of amulti-parameter determination.
 10. The method of claim 9, wherein saidmethod is carried out as part of a multi-parameter determination withthe use of chip technology and/or as part of an automated method withcomputer-assisted evaluation of the measured results.
 11. The method ofclaim 10, wherein, in the multi-parameter determination, at least onefurther parameter relevant for sepsis diagnosis is determined.
 12. Themethod of claim 11, wherein the further parameter or parameters relevantfor the sepsis diagnosis is or are selected from the group whichconsists of total procalcitonin, procalcitonin 3-116, CA 125, CA 19-9,S100B, S100A proteins, LASP-1, soluble cytokeratin fragments, inparticular CYFRA 21, TPS and/or soluble cytokeratin-1 fragments (sCY1F),the peptides inflammin and CHP, the prohormones pro-ANP, pro-BNP,pro-adrenomedullin, pro-endothelin, pro-vasopressin and their fragmentsusable for diagnostic purposes, glycine N-acyl transferase (GNAT),carbamoyl phosphate synthetase 1 (CPS 1), the C-reactive protein (CRP)and fragments thereof and of cytokines.
 13. An isolated antibody whichbinds selectively only to the intact procalcitonin 1 to 116 (SEQ IDNO: 1) or procalcitonin derivatives which have a procalcitonin aminoacid sequence or procalcitonin amino acid partial sequence with at leastone of the amino acids 1 and 2, preferably with both amino acids 1 and2, of the amino terminus of the sequence of the complete procalcitonin 1to 116 (SEQ ID NO: 1).
 14. The isolated antibody of claim 13, which isobtainable by immunisation of an animal with an antigen, in the form ofa conjugate with a peptide which is or comprises a peptide according toSEQ ID NO:
 4. 15. The isolated antibody of claim 13, wherein saidantibody is a monoclonal antibody.
 16. A kit for carrying out a methodfor measuring procalcitonin and procalcitonin derivatives, wherein saidkit comprises: at least one antibody which binds selectively only to theintact procalcitonin 1 to 116 (SEQ, ID NO: 1) or procalcitoninderivatives which have a procalcitonin amino acid sequence orprocalcitonin amino acid partial sequence with at least one of the aminoacids 1 and 2, preferably with both amino acids 1 and 2, of the aminoterminus of the sequence of the complete procalcitonin 1 to 116 (SEQ IDNO: 1) bound to a solid phase or in marked or selectively markable formand a competitor or a standard compound in the form of a peptide whichcomprises at least amino acids 1 to 6 of the amino acid sequence of thecomplete procalcitonin 1 to 116 (SEQ ID NO: 1) and which is selectivelybound by the at least one antibody.
 17. The method of claim 16, whereinthe competitor is bound to a solid phase or is marked or selectivelymarkable.
 18. A kit for measuring procalcitonin and procalcitoninderivatives, said kit comprising: a first antibody which bindsselectively only to the intact procalcitonin 1 to 116 (SEQ ID NO: orprocalcitonin derivatives which have a procalcitonin amino acid sequenceor procalcitonin amino acid partial sequence with at least one of theamino acids 1 and 2, preferably with both amino acids 1 and 2, of theamino terminus of the sequence of the complete procalcitonin 1 to 116(SEQ ID NO: 1), and a second antibody which selectively binds to anydesired amino acid partial sequence of the complete procalcitonin 1 to116 (SEQ ID NO: 1) which does not comprise the amino acids 1 and 2 ofthe amino acid terminus of procalcitonin.
 19. The kit according to ofclaim 18, wherein one of the two antibodies is bound to a solid phaseand the other antibody is marked or selectively markable.
 20. The kit ofclaim 18, wherein both antibodies are present in a form suitable for useas a dispersion in a liquid phase, a first marking component which ispart of a marking system based on fluorescence or chemiluminescenceextinction or amplification being bound to the first antibody, and thesecond marking component of this marking system being bound to thesecond antibody, so that, after binding of the two antibodies to thepeptide fragment to be detected, a measurable signal which permitsdetection of the resulting sandwich complexes in the measuring solutionis produced.
 21. The kit of claim 20, the marking system comprising rareearth cryptates or chelates in combination with a fluorescence orchemiluminescence dye, in particular of the cyanine type.
 22. The kit ofclaim 14, wherein the kit also comprises a compound for stabilizing thebiological sample by inhibiting enzymatic action ofdipeptidyl-aminopeptidase IV (DAP IV; DP IV; CD 26).